PROJECT SUMMARY Normal cells are dependent upon the extracellular cell matrix (ECM) for survival, and undergo apoptosis when they lose contact with the ECM ? a phenomenon termed anoikis. The acquisition of anoikis resistance is a critical step that contributes prominently to the metastatic progression in ovarian cancer. However, the factors and regulatory pathways that confer anoikis resistance in ovarian cancer remain largely unknown. Thus, to identify factors that confer anoikis resistance, we performed an unbiased druggable genome-based RNAi screen and identified ATAD2 as a novel factor that confers anoikis resistance in high-grade serous ovarian cancer (HGS- OvCa) cells. Additionally, we document that ATAD2 is overexpressed in HGS-OvCa patient samples and its overexpression predicts significantly reduced overall survival (OS) and progression-free survival (PFS). Our preliminary data that makes the basis of this research proposal provides strong evidence supporting the scientific premise that ATAD2 is necessary for anoikis resistance and thus is a potential driver of HGS-OvCa metastasis. The overall objective for this research proposal is to determine the in vivo role of ATAD2 in facilitating HGS- OvCa metastasis, understand its mechanism-of-action and evaluate in vivo pharmacological targeting of ATAD2 for metastatic HGS-OvCa therapy. Specifically, Aim 1 experiments will determine the role of ATAD2 as a driver of HGS-OvCa metastasis and evaluate it as a drug target for metastatic HGS-OvCa cancer therapy. We will use both genetic approach and highly-effective and selective ATAD2 small molecule inhibitor BAY-850-based pharmacological approach for achieving the goals of Aim 1. For genetic approach, we will use complementary organ-specific and spontaneous metastasis models based on orthotopic xenograft of HGS-OvCa cells. For pharmacological approach, in addition to organ-specific and spontaneous mouse models of HGS-OvCa metastasis, we will also use a novel humanized HGS-OvCa xenograft model with intact innate and adoptive human immune system. Aim 2, experiments will determine the mechanism by which ATAD2 confers anoikis resistance and promote HGS-OvCa metastasis. In preliminary studies, we found that ATAD2 represses the expression of pro-apoptotic gene BAD, which is necessary for ATAD2 inhibition-induced anoikis resistance. Based on these results, we will ascertain the role of BAD as a downstream mediator of ATAD2-indued anoikis resistance and HGS-OvCa metastasis. The experimental approaches will utilize biochemical, genetic, cell culture-based methods and in vivo mouse model of anoikis resistance and HGS-OvCa metastasis. In particular, we will use organ-specific and spontaneous metastasis model based on orthotopic HGS-OvCa cells transplant. Collectively, these results will identify a novel druggable dependency pathway that via promoting anoikis resistance facilitates HGS-OvCa metastasis, and thus can be targeted for effectively treating highly aggressive metastatic HGS-OvCa.